Examining the stability of viral RNA and DNA in wastewater: Effects of storage time, temperature, and freeze-thaw cycles

•S gene of SARS-Cov-2 is more sensitive to freeze-thaw than other regions tested.•Phi6 is a stable surrogate for SARS-CoV-2 under different storage conditions.•Freeze-thawing raw wastewater negatively impacts next-generation sequencing metrics.•Historical (12–16 months) wastewater samples stored at −80 °C maintained integrity. Wastewater-based epidemiology (WBE) has been demonstrably successful as a relatively unbiased tool for monitoring levels of SARS-CoV-2 virus circulating in communities during the COVID-19 pandemic. Accumulated biobanks of wastewater samples allow retrospective exploration of spatial and temporal trends for public health indicators such as chemicals, viruses, antimicrobial resistance genes, and the possible emergence of novel human or zoonotic pathogens. We investigated virus resilience to time, temperature, and freeze-thaw cycles, plus the optimal storage conditions to maintain the stability of genetic material (RNA/DNA) of viral +ssRNA (Envelope – E, Nucleocapsid – N and Spike protein – S genes of SARS-CoV-2), dsRNA (Phi6 phage) and circular dsDNA (crAssphage) in wastewater. Samples consisted of (i) processed and extracted wastewater samples, (ii) processed and extracted distilled water samples, and (iii) raw, unprocessed wastewater samples. Samples were stored at –80 °C, –20 °C, 4 °C, or 20 °C for 10 days, going through up to 10 freeze-thaw cycles (once per day). Sample stability was measured using reverse transcription quantitative PCR, quantitative PCR, automated electrophoresis, and short-read whole genome sequencing. Exploring different areas of the SARS-CoV-2 genome demonstrated that the S gene in processed and extracted samples showed greater sensitivity to freeze-thaw cycles than the E or N genes. Investigating surrogate and normalisation viruses showed that Phi6 remains a stable comparison for SARS-CoV-2 in a laboratory setting and crAssphage was relatively resilient to temperature variation. Recovery of SARS-CoV-2 in raw unprocessed samples was significantly greater when stored at 4 °C, which was supported by the sequencing data for all viruses – both time and freeze-thaw cycles negatively impacted sequencing metrics. Historical extracts stored at –80 °C that were re-quantified 12, 14 and 16 months after original quantification showed no major changes. This study highlights the importance of the fast processing and extraction of wastewater samples, following which viruses are relatively robust to storage at a range of temp